A DNA circuit that records molecular events.

Characterizing the relative onset time, strength, and duration of molecular signals is critical for understanding the operation of signal transduction and genetic regulatory networks. However, detecting multiple such molecules as they are produced and then quickly consumed is challenging. A MER can encode information about transient molecular events as stable DNA sequences and are amenable to downstream sequencing or other analysis. Here, we report the development of a de novo molecular event recorder that processes information using a strand displacement reaction network and encodes the information using the primer exchange reaction, which can be decoded and quantified by DNA sequencing. The event recorder was able to classify the order at which different molecular signals appeared in time with 88% accuracy, the concentrations with 100% accuracy, and the duration with 75% accuracy. This simultaneous and highly programmable multiparameter recording could enable the large-scale deciphering of molecular events such as within dynamic reaction environments, living cells, or tissues.

Aptamer Technologies in Neuroscience, Neuro-Diagnostics and Neuro-Medicine Development.

Aptamers developed using in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology are single-stranded nucleic acids 10-100 nucleotides in length. Their targets, often with specificity and high affinity, range from ions and small molecules to proteins and other biological molecules as well as larger systems, including cells, tissues, and animals. Aptamers often rival conventional antibodies with improved performance, due to aptamers' unique biophysical and biochemical properties, including small size, synthetic accessibility, facile modification, low production cost, and low immunogenicity. Therefore, there is sustained interest in engineering and adapting aptamers for many applications, including diagnostics and therapeutics. Recently, aptamers have shown promise as early diagnostic biomarkers and in precision medicine for neurodegenerative and neurological diseases. Here, we critically review neuro-targeting aptamers and their potential applications in neuroscience research, neuro-diagnostics, and neuro-medicine. We also discuss challenges that must be overcome, including delivery across the blood-brain barrier, increased affinity, and improved in vivo stability and in vivo pharmacokinetic properties.

Enzyme Core Spherical Nucleic Acid That Enables Enhanced Cuproptosis and Antitumor Immune Response through Alleviating Tumor Hypoxia.

Cuproptosis, a copper-dependent cell death process, has been confirmed to further activate the immune response and mediate the immune resistance. However, hypoxic tumor microenvironment hampers cuproptosis sensitivity and suppresses the body's antitumor immune response. Herein, we have successfully immobilized and functionalized catalase (CAT) with long single-stranded DNA containing polyvalent CpG sequences through rolling circle amplification (RCA) techniques, obtaining an enzyme-cored spherical nucleic acid nanoplatform (CAT-ecSNA-Cu) to deliver copper ions for cuproptosis. The presence of long-stranded DNA-protected CAT enhances mitochondrial respiration by catalyzing the conversion of H2O2 to O2, thereby sensitizing cuproptosis. Meanwhile, increased tumor oxygenation suppresses the expression of the hypoxia-inducible factor-1 (HIF-1) protein, resulting in the alleviation of the immunosuppressive tumor microenvironment. Of note, cuproptosis induces immunogenic cell death (ICD), which facilitates dendritic cell (DC) maturation and enhances antigen presentation through polyCpG-supported Toll-like receptor 9 (TLR9) activation. Furthermore, cuproptosis-induced PD-L1 upregulation in tumor cells complements checkpoint blockers (αPD-L1), enhancing antitumor immunity. The strategy of enhancing cuproptosis-mediated antitumor immune responses by alleviating hypoxia effectively promotes the activation and proliferation of effector T cells, ultimately leading to long-term immunity against cancer.

Domain-Targeted Membrane Partitioning of Specific Proteins with DNA Nanodevices.

The cell membrane exhibits a remarkable complexity of lipids and proteins that dynamically segregate into distinct domains to coordinate various cellular functions. The ability to manipulate the partitioning of specific membrane proteins without involving genetic modification is essential for decoding various cellular processes but highly challenging. In this work, by conjugating cholesterols or tocopherols at the three bottom vertices of the DNA tetrahedron, we develop two sets of nanodevices for the selective targeting of lipid-order (Lo) and lipid-disorder (Ld) domains on the live cell membrane. By incorporation of protein-recognition ligands, such as aptamers or antibodies, through toehold-mediated strand displacement, these DNA nanodevices enable dynamic translocation of target proteins between these two domains. We first used PTK7 as a protein model and demonstrated, for the first time, that the accumulation of PTK7 to the Lo domains could promote tumor cell migration, while sequestering it in the Ld domains would inhibit the movement of the cells. Next, based on their modular nature, these DNA nanodevices were extended to regulate the process of T cell activation through manipulating the translocation of CD45 between the Lo and the Ld domains. Thus, our work is expected to provide deep insight into the study of membrane structure and molecular interactions within diverse cell signaling processes.

Engineering a Dual-Receptor Targeted Multivalent Probe for Enhanced Magnetic Resonance Imaging of Metastatic Cancer.

Noninvasive monitoring of cancer metastasis is essential to improving clinical outcomes. Molecular MRI (mMRI) is a special implementation of noninvasive molecular imaging that promises to offer a powerful means for early detection and analysis of pathological states of cancer by tracking molecular markers. However, this is often hindered by the challenging issue of obtaining transformable mMRI contrast agents with high sensitivity, specificity, and broad applicability, given the high tumor heterogeneity and complex metastatic features. Herein, we present a dual-receptor targeted, multivalent recognition strategy and report a new class of mMRI probes for enhanced imaging of metastatic cancer. This probe is designed by covalently conjugating Gd-chelate with phenylboronic acid and an aptamer via an affordable polymerization chemistry to concurrently target two different cell-membrane receptors that are commonly overexpressed and highly implicated in both tumorigenesis and metastasis. Moreover, the polymerization chemistry allows the probe to contain a bunch of targeting ligands and signal reporters in a single chain, which not only leads to more than 2-fold enhancement in T1 relaxivity at 1.5 T compared to the commercial contrast agent but also enables it to actively target tumor cells in a multivalent recognition manner, contributing to a much higher imaging contrast than single-receptor targeted probes and the commercial agent in mouse models with lung metastases, yet without inducing systemic side effects. We expect this study to offer a useful molecular tool to promote transformable applications of mMRI and a better understanding of molecular mechanisms involved in cancer development.

Multivalent Aptamer-Based Lysosome-Targeting Chimeras (LYTACs) Platform for Mono- or Dual-Targeted Proteins Degradation on Cell Surface.

Selective protein degradation platforms have opened novel avenues in therapeutic development and biological inquiry. Antibody-based lysosome-targeting chimeras (LYTACs) have emerged as a promising technology that extends the scope of targeted protein degradation to extracellular targets. Aptamers offer an advantageous alternative owing to their potential for modification and manipulation toward a multivalent state. In this study, a chemically engineered platform of multivalent aptamer-based LYTACs (AptLYTACs) is established for the targeted degradation of either single or dual protein targets. Leveraging the biotin-streptavidin system as a molecular scaffold, this investigation reveals that trivalently mono-targeted AptLYTACs demonstrate optimum efficiency in degrading membrane proteins. The development of this multivalent AptLYTACs platform provides a principle of concept for mono-/dual-targets degradation, expanding the possibilities of targeted protein degradation.

Enhancing Specific Fluorescence In Situ Hybridization with Quantum Dots for Single-Molecule RNA Imaging in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Single-molecule fluorescence in situ hybridization (smFISH) represents a promising approach for the quantitative analysis of nucleic acid biomarkers in clinical tissue samples. However, low signal intensity and high background noise are complications that arise from diagnostic pathology when performed with smFISH-based RNA imaging in formalin-fixed paraffin-embedded (FFPE) tissue specimens. Moreover, the associated complex procedures can produce uncertain results and poor image quality. Herein, by combining the high specificity of split DNA probes with the high signal readout of ZnCdSe/ZnS quantum dot (QD) labeling, we introduce QD split-FISH, a high-brightness smFISH technology, to quantify the expression of mRNA in both cell lines and clinical FFPE tissue samples of breast cancer and lung squamous carcinoma. Owing to its high signal-to-noise ratio, QD split-FISH is a fast, inexpensive, and sensitive method for quantifying mRNA expression in FFPE tumor tissues, making it suitable for biomarker imaging and diagnostic pathology.

Unlocking the Potential of Pterostilbene: A Pharmaceutical Element for Aptamer-Based Nanomedicine.

Natural compounds like pterostilbene (PTE) have gained recognition for their various biological activities and potential health benefits. However, challenges related to bioavailability and limited clinical efficacy have prompted efforts to strengthen their therapeutic potential. To meet these challenges, we herein rationally designed and successfully synthesized a pharmaceutical phosphoramidite that allows for the programmable incorporation of PTE into oligonucleotides. The resultant aptamer-PTE conjugate can selectively bind to cancer cells, leading to a specific internalization and drug release. Moreover, compared with free PTE, the conjugate exhibits superior cytotoxicity in cancer cells. Specifically, in a zebrafish xenograft model, the nanomedicine effectively inhibits tumor growth and neovascularization, highlighting its potential for targeted antitumor therapy. This approach presents a promising avenue for harnessing the therapeutic potential of natural compounds via a nanomedicine solution.

Artificial DNA Framework Channel Modulates Antiapoptotic Behavior in Ischemia-Stressed Cells via Destabilizing Promoter G-Quadruplex.

Regulating folding/unfolding of gene promoter G-quadruplexes (G4s) is important for understanding the topological changes in genomic DNAs and the biological effects of such changes on important cellular events. Although many G4-stabilizing ligands have been screened out, effective G4-destabilizing ligands are extremely rare, posing a great challenge for illustrating how G4 destabilization affects gene function in living cells under stress, a long-standing question in neuroscience. Herein, we report a distinct methodology able to destabilize gene promoter G4s in ischemia-stressed neural cells by mitigating the ischemia-induced accumulation of intracellular K+ with an artificial membrane-spanning DNA framework channel (DFC). We also show that ischemia-triggered K+ influx is positively correlated to anomalous stabilization of promoter G4s and downregulation of Bcl-2, an antiapoptotic gene with neuroprotective effects against ischemic injury. Intriguingly, the DFC enables rapid transmembrane transport of excessive K+ mediated by the internal G4 filter, leading to the destabilization of endogenous promoter G4 in Bcl-2 and subsequent turnover of gene expression at both transcription and translation levels under ischemia. Consequently, this work enriches our understanding of the biological roles of endogenous G4s and may offer important clues to study the cellular behaviors in response to stress.

Exosome Heterogeneity Affects the Distal "Barrier-Crossing" Trafficking of Exosome Encapsulated Quantum Dots.

The biological activities of nanoparticles (NPs), which include endocytosis by macrophages and subsequent intracellular degradation and/or release, transfer to other cells, or translocation across tissue barriers, highly depend on their fate in living organisms. Yet, translocation across barriers, especially the distal "barrier-crossing" trafficking of NPs, is still unclear. The exosome (Exo) plays a crucial role in intercellular communication and biological barrier trafficking. Here, we report that ZnCdSe@ZnS quantum dots (QDs), as a representation of NPs in biomedical applications, could cross the blood-brain barrier and approach the mouse brain via active Exo encapsulation. By employing multiple techniques, we demonstrated that QDs were internalized by macrophages (J774A.1) and tumor cells (HeLa) and then released to the extracellular environment along with Exo. Exo encapsulation facilitates the distal barrier-crossing trafficking of QDs in vivo, while Exo biogenesis inhibitor GW4869 suppressed the QDs enriched in the brains of mice with a 4T1-Luc breast cancer xenograft. Interestingly, Exo heterogeneity affects the distal trafficking of enveloped QDs. Exo derived from tumorous HeLa cells, not macrophages, that were enriched in functional proteins with cell adhesion, cell migration, axon guidance, and cell motility, showed a better capacity for the remote trafficking of QDs. This study proposes Exo as a vehicle to deliver exogenous NPs to translocate across the distal barrier and provides further information for biomedical application and the risk assessment of NPs.

Toxicological evaluation and concentration of airborne PM0.1 in high air pollution period in Guangzhou, China.

The emissions and exposure limits for airborne PM0.1 are lacking, with limited scientific data for toxicity. Therefore, we continuously monitored and calculated the number and mass concentrations of airborne PM0.1 in December 2017, January 2018 and March 2018 during the high pollution period in Guangzhou. We collected PM0.1 from the same period and analyzed their chemical components. A549, THP-1 and A549/THP-1 co-cultured cells were selected for exposure to PM0.1, and evaluated for toxicological responses. Our aims are to 1) measure and analyze the number and mass concentrations, and chemical components of PM0.1; 2) evaluate and compare PM0.1 toxicity to different airway cells models at different time points. Guangzhou had the highest mass concentration of PM0.1 in December 2017, while the number concentration was the lowest. Chemical components in PM0.1 vary significantly at different time periods, and the correlation between the chemical composition or source of PM0.1 and the mass and number concentration of PM0.1 was dissimilar. Exposure to PM0.1 disrupted cell membranes, impaired mitochondrial function, promoted the expression of inflammatory mediators, and interfered with DNA replication in the cell cycle. The damage caused by exposure to PM0.1 at different times exhibited variations across different types of cells. PM0.1 in March 2018 stimulated co-cultured cells to secrete more inflammatory mediators, and CMA was significantly related to the expression of them. Our study indicates that it is essential to monitor both the mass and number concentrations of PM0.1 throughout all seasons annually, as conventional toxicological experiments and the internal components of PM0.1 may not effectively reveal the health damages caused by elevated number levels of PM0.1.

Dual-responsive 3D DNA nanomachines cascaded hybridization chain reactions for novel self-powered flexible microRNA-detecting platform.

The microRNA-21 is closely related to chromatin remodeling and epigenetic regulation. In this work, an efficient double-response 3D DNA nanomachine (DRDN) was assembled by co-immobilizing two different lengths of hairpin DNA on the surface of gold nanoparticles (AuNPs) to capture microRNA-21 (miRNA-21), recycle miRNA-21, and trigger hybridization chain reactions (HCR). This work reports the fabrication of a laser-scribed graphene (LSG) electrode with excellent flexibility and electrical conductivity by laser-scribing commercial polyimide films (PI). The as-proposed self-powered biosensing platform presents significantly increased instantaneous current to in real-time monitor miRNA-21 by a capacitor. The biosensing platform exhibited highly sensitive detection of miRNA-21 with a detection limit of 0.142 fM in the range of 0.5 fM to 1 × 104 fM, and demonstrated high efficiency in the analysis of the tumor markers.

Aptamer-Based Multiparameter Analysis for Molecular Profiling of Hematological Malignancies.

The subtypes of hematological malignancies (HM) with minimal molecular profile differences display an extremely heterogeneous clinical course and a discrepant response to certain treatment regimens. Profiling the surface protein markers offers a potent solution for precision diagnosis of HM by differentiating among the subtypes of cancer cells. Herein, we report the use of Cell-SELEX technology to generate a panel of high-affinity aptamer probes that are able to discriminate subtle differences among surface protein profiles between different HM cells. Experimental results show that these aptamers with apparent dissociation constants (Kd) below 10 nM display a unique recognition pattern on different HM subtypes. By combining a machine learning model on the basis of partial least-squares discriminant analysis, 100% accuracy was achieved for the classification of different HM cells. Furthermore, we preliminarily validated the effectiveness of the aptamer-based multiparameter analysis strategy from a clinical perspective by accurately classifying complex clinical samples, thus providing a promising molecular tool for precise HM phenotyping.

Functional Selection of Tau Oligomerization-Inhibiting Aptamers.

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3ß-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

Association Between Home Renovation and Sleeping Problems Among Children Aged 6-18 Years: A Nationwide Survey in China.

BACKGROUND: Although the indoor environment has been proposed to be associated with childhood sleep health, to our knowledge no study has investigated the association between home renovation and childhood sleep problems. METHODS: The study included 186,470 children aged 6-18 years from the National Chinese Children Health Study (2012-2018). We measured childhood sleeping problems via the Chinese version of the Sleep Disturbance Scale for Children (C-SDSC). Information on home renovation exposure within the recent 2 years was collected via parent report. We estimated associations between home renovation and various sleeping problems, defined using both continuous and categorized (binary) C-SDSC t-scores, using generalized mixed models. We fitted models with city as a random effect variable, and other covariates as fixed effects. RESULTS: Out of the overall participants, 89,732 (48%) were exposed to recent home renovations. Compared to the unexposed group, children exposed to home renovations had higher odds of total sleep disorder (odd ratios [OR] = 1.3; 95% confidence interval [CI] = 1.2, 1.4). Associations varied when we considered different types of home renovation materials. Children exposed to multiple types of home renovation had higher odds of sleeping problems. We observed similar findings when considering continuous C-SDSC t-scores. Additionally, sex and age of children modified the associations of home renovation exposure with some of the sleeping problem subtypes. CONCLUSIONS: We found that home renovation was associated with higher odds of having sleeping problems and that they varied when considering the type of renovation, cumulative exposure, sex, and age differences.

Serum Protein Fishing for Machine Learning-Boosted Diagnostic Classification of Small Nodules of Lung.

Diagnosis of benign and malignant small nodules of the lung remains an unmet clinical problem which is leading to serious false positive diagnosis and overtreatment. Here, we developed a serum protein fishing-based spectral library (ProteoFish) for data independent acquisition analysis and a machine learning-boosted protein panel for diagnosis of early Non-Small Cell Lung Cancer (NSCLC) and classification of benign and malignant small nodules. We established an extensive NSCLC protein bank consisting of 297 clinical subjects. After testing 5 feature extraction algorithms and six machine learning models, the Lasso algorithm for a 15-key protein panel selection and Random Forest was chosen for diagnostic classification. Our random forest classifier achieved 91.38% accuracy in benign and malignant small nodule diagnosis, which is superior to the existing clinical assays. By integrating with machine learning, the 15-key protein panel may provide insights to multiplexed protein biomarker fishing from serum for facile cancer screening and tackling the current clinical challenge in prospective diagnostic classification of small nodules of the lung.

Topological Single-stranded DNA Encoding and Programmable Assembly of Molecular Nanostructures for NIR-II Cancer Theranostics.

Molecular nanotechnology promises to offer privileged access to developing NIR-II materials with precise structural and functional manipulation for transformable theranostic applications. However, the lack of an affordable, yet general, method makes this goal currently inaccessible. By virtue of the intriguing nucleic acid chemistry, here we present an artificial base-directed topological single-strand DNA encoding design that enables one-step synthesis of valence-controlled NIR-II molecular nanostructures and spatial assembly of these nanostructures to modulate their behaviors in living systems. As proof-of-concept studies, we construct ultrasmall Ag2 S quantum dots and pH-responsive, size-tunable CuS assemblies for in vivo NIR-II fluorescence imaging and deep tumor photothermal therapy. This work paves a new way for creating functionally diversified architectures and broadens the scope of DNA-encoded material engineering.

An Aptamer-Based Nanoflow Cytometry Method for the Molecular Detection and Classification of Ovarian Cancers through Profiling of Tumor Markers on Small Extracellular Vesicles.

Molecular profiling of protein markers on small extracellular vesicles (sEVs) is a promising strategy for the precise detection and classification of ovarian cancers. However, this strategy is challenging owing to the lack of simple and practical detection methods. In this work, using an aptamer-based nanoflow cytometry (nFCM) detection strategy, a simple and rapid method for the molecular profiling of multiple protein markers on sEVs was developed. The protein markers can be easily labeled with aptamer probes and then rapidly profiled by nFCM. Seven cancer-associated protein markers, including CA125, STIP1, CD24, EpCAM, EGFR, MUC1, and HER2, on plasma sEVs were profiled for the molecular detection and classification of ovarian cancers. Profiling these seven protein markers enabled the precise detection of ovarian cancer with a high accuracy of 94.2 %. In addition, combined with machine learning algorithms, such as linear discriminant analysis (LDA) and random forest (RF), the molecular classifications of ovarian cancer cell lines and subtypes were achieved with overall accuracies of 82.9 % and 55.4 %, respectively. Therefore, this simple, rapid, and non-invasive method exhibited considerable potential for the auxiliary diagnosis and molecular classification of ovarian cancers in clinical practice.

Regulation of Cellular Signaling with an Aptamer Inhibitor to Impede Cancer Metastasis.

Tumor invasion and metastasis are the main causes of tumor progression and are the leading causes of death among cancer patients. In the present study, we propose a strategy to regulate cellular signaling with a tumor metastasis-relevant cytoskeleton-associated protein 4 (CKAP4) specific aptamer for the achievement of tumor metastasis inhibition. The designed aptamer could specifically bind to CKAP4 in the cell membranes and cytoplasm to block the internalization and recycling of α5ß1 integrin, resulting in the disruption of the fibronectin-dependent cell adhesion and the weakening of the cell traction force. Moreover, the aptamer is able to impede the interaction between CKAP4 and Dickkopf1 (DKK1) to further block the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, which subsequently reduces AKT phosphorylation and inhibits the reorganization of the actin cytoskeleton in cell migration. The synergetic function of the designed aptamer in inhibiting cancer cell adhesion and blocking the PI3K signaling pathway enables efficient tumor cell metastasis suppression. The aptamer with specific targeting ability in regulating cellular signaling paves the way for cancer treatment and further provides a guiding ideology for inhibiting tumor metastasis.

Diallyl disulfide synergizes with melphalan to increase apoptosis and DNA damage through elevation of reactive oxygen species in multiple myeloma cells.

Diallyl disulfide (DADS), one of the main components of garlic, is well known to have anticancer effects on multiple cancers. However, its efficacy in treating multiple myeloma (MM) is yet to be determined. We explored the effects of DADS on MM cells and investigated the synergistic effects of DADS when combined with five anti-MM drugs, including melphalan, bortezomib, carfilzomib, doxorubicin, and lenalidomide. We analyzed cell viability, cell apoptosis, and DNA damage to determine the efficacy of DADS and the drug combinations. Our findings revealed that DADS induces apoptosis in MM cells through the mitochondria-dependent pathway and increases the levels of γ-H2AX, a DNA damage marker. Combination index (CI) measurements indicated that the combination of DADS with melphalan has a significant synergistic effect on MM cells. This was further confirmed by the increases in apoptotic cells and DNA damage in MM cells treated with the two drug combinations compared with those cells treated with a single drug alone. The synergy between DADS and melphalan was also observed in primary MM cells. Furthermore, mechanistic investigations showed that DADS decreases reduced glutathione (GSH) levels and increases reactive oxygen species (ROS) production in MM cells. The addition of GSH is effective in neutralizing DADS cytotoxicity and inhibiting the synergy between DADS and melphalan in MM cells. Taken together, our study highlights the effectiveness of DADS in treating MM cells and the promising therapeutic potential of combining DADS and melphalan for MM treatment.